Vesicular Stomatitis Virus Matrix Protein Inhibits Host Cell Gene Expression by Targeting the Nucleoporin Nup98

Molecular Cell, Vol. 6, 1243–1252, November, 2000, Copyright 2000 by Cell Press

Vesicular Stomatitis Virus Matrix Protein Inhibits Host Cell Gene Expression by Targeting the Nucleoporin Nup98 Cayetano von Kobbe,*k Jan M. A. van Deursen,† Joa˜o P. Rodrigues,‡ Delphine Sitterlin,* Angela Bachi,* Xiaosheng Wu,† Matthias Wilm,* Maria Carmo-Fonseca,‡ and Elisa Izaurralde*§ * European Molecular Biology Laboratory Meyerhofstrasse 1 D-69117 Heidelberg Germany † Mayo Clinic Rochester, Minnesota 55905 ‡ Institute of Histology and Embryology Faculty of Medicine University of Lisbon 1699 Lisboa Codex Portugal

Summary Vesicular stomatitis virus matrix protein (VSV M) has been shown to inhibit both transcription and nucleocytoplasmic transport. We have isolated a mutant form of M, termed M(D), lacking both inhibitory activities. HeLa cells expressing M, but not M(D), accumulate polyadenylated RNAs within the nucleus. Concomitantly, a fraction of M, but not of the M(D) mutant, localizes at the nuclear rim. Additionally, the nucleoporin Nup98 specifically interacts with M but not with M(D). In Nup98⫺/⫺ cells, both the levels of M at the nuclear envelope and its inhibitory effects on host cell– directed expression of reporter genes were significantly reduced. Together, our data demonstrate that VSV M inhibits host cell gene expression by targeting a nucleoporin and primarily blocking nuclear export. Introduction The vesicular stomatitis virus (VSV) belongs to the Rhabdoviridiae family and causes acute infections in a wide range of mammalian hosts (reviewed by Wagner and Rose, 1995). Its matrix protein (VSV M) has been shown to play a key role in virus assembly and budding and in the inhibition of host cell gene expression during infection. When expressed in vertebrate cells, in the absence of any other viral protein, VSV M produces dramatic alterations in cellular RNA metabolism and mRNA expression (Black and Lyles, 1992; Ahmed and Lyles, 1998; and references therein). Among its pleiotropic effects, VSV M has been shown to shut off transcription by host RNA polymerases I and II (Ahmed and Lyles, 1998). Furthermore, when expressed in Xenopus laevis oocytes, VSV M inhibited nuclear export of U snRNA, rRNA, 5S § To whom correspondence should be addressed (e-mail: izaurralde@

embl-heidelberg.de). k Present address: Laboratory of Molecular Genetics, NIH, Baltimore, Maryland 21224.

rRNA, and mRNA without affecting the export of tRNA molecules (Her et al., 1997). Nuclear transport occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors that shuttle between the nucleus and cytoplasm. Most of the transport receptors identified to date are members of a large family of RanGTP binding proteins exhibiting limited sequence similarity with the Ran binding domain of importin-␤, and have been termed importins/exportins or karyopherins. The interaction of these receptors with their cargoes or with nucleoporins is regulated by the small GTPase, Ran (reviewed by Nakielny and Dreyfuss, 1999). In vertebrates, export of U snRNA, tRNA, and 5S rRNA is mediated by members of the importin-␤-like family of receptors. tRNA export is mediated by exportin-t, which directly binds to the tRNA molecules in the presence of RanGTP (Arts et al., 1998; Kutay et al., 1998). CRM1 is the export receptor for polymerase II-transcribed spliceosomal U snRNA (Fornerod et al., 1997) and has also been implicated in the export of 5S rRNA (Fischer et al., 1995). Export of cellular mRNAs might not be mediated by any member of the importin-␤-related receptor family (reviewed by Nakielny and Dreyfuss, 1999). The NPCassociated proteins TAP/Mex67p and RAE1/Gle2p have been proposed to fulfill the role of export receptors for mRNA (Bachi et al., 2000). The different RNP export pathways converge at the NPC. The overall three-dimensional architecture of the NPC is evolutionarily conserved and consists of three structural units: a ring-like central framework embracing the central channel of the pore is sandwiched between the cytoplasmic ring, from which eight cytoplasmic fibrils emanate, and the nuclear ring, which anchors the nuclear basket (Yang et al., 1998; Stoffler et al., 1999). The structural units of the NPC are composed of multiple copies of up to about 50 polypeptides called nucleoporins. Nucleoporins often contain multiple phenylalanineglycine dipeptide repeats (FG) clustered in domains, which in vertebrates are glycosylated by addition of O-linked N-acetylglucosamine (GlcNAc) (reviewed by Rout and Wente, 1994). Evidence of a role for vertebrate nucleoporins in RNA export came from the use of anti-nucleoporin antibodies and of wheat germ agglutinin (WGA), a lectin that binds to O-linked GlcNAc residues. WGA or antibodies that recognize the FG repeat region of various nucleoporins block several export processes including those of U snRNA, 5S rRNA, and tRNA (Featherstone et al., 1988; Neuman de Vegvar and Dahlberg, 1990; Terns and Dahlberg, 1994). These reagents bind to a common subset of glycosylated, FG repeat–containing nucleoporins such as p62, Nup98, and CAN. The role of specific nucleoporins in export has been analyzed by using specific antibodies and by overexpression of nucleoporin domains in cultured cells. Microinjection of antibodies raised against the nucleoporin Nup98 in Xenopus oocytes inhibited U snRNA and mRNA export without affecting tRNA export (Powers et al., 1997), whereas overexpression of Nup153 in mammalian cells resulted in

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nuclear accumulation of polyadenylated RNA (Bastos et al., 1997). To investigate the mechanism of nuclear export inhibition by VSV M, we first mapped the inhibitory domain of M protein to residues found between positions 44 and 77. Within this domain, substitution of residues 52–54 by alanines resulted in a mutant form of the protein M(D), lacking the export inhibitory activity. HeLa cells expressing M protein, but not the M(D) mutant, accumulate polyadenylated RNA within the nucleus. At the same time, a fraction of wild-type protein, but not of the M(D) mutant, localizes at the nuclear rim. We identify the nucleoporin Nup98 as a cellular protein able to discriminate between wild-type M and the M(D) mutant. Experiments performed in Nup98-deficient embryonic mouse cells confirmed that Nup98 represents the major binding site for VSV M at the NPC. Furthermore, in these cells VSV M–mediated inhibition of the host cell transcription was dramatically impaired. Together, our data indicate that VSV M acts to deregulate nucleocytoplasmic transport, and thereby cellular gene expression, by targeting Nup98. Results Inhibition of RNA Nuclear Export by VSV M Is Mediated by Its N-Terminal Domain To identify the cellular factor(s) that mediate VSV M export inhibition, we first defined the domain of the protein required for its inhibitory activity by microinjection into Xenopus oocytes. M is a basic protein of 229 amino acids consisting of two domains: the amino-terminal domain (residues 1–43) and a trypsin-resistant core (residues 44–229) (Figure 2A; Lenard and Vanderoef, 1990; and references therein). Preliminary experiments showed that the trypsin-resistant core of M inhibited RNA nuclear export as efficiently as the full-length protein (data not shown). To define more precisely the M inhibitory domain, we generated truncated versions of the protein. M and its deletion mutants were expressed in E. coli, purified, and injected into Xenopus oocyte nuclei, together with a mixture of labeled RNAs. This mixture consisted of DHFR mRNA, histone H4 mRNA, U1⌬Sm, U5⌬Sm, U6⌬ss, and the human initiator methionyl tRNA. Export of U1⌬Sm and U5⌬Sm snRNAs is inhibited by M (Her et al., 1997). Therefore, U snRNAs served as controls for the inhibitory activity of the recombinant proteins. U6⌬ss RNA is not exported from the nucleus and served as a control for the accuracy of the nuclear injections (Vankan et al., 1992). Immediately after injection, all RNAs were found in the nuclear fraction (Figure 1, lanes 1–3 in all panels). Following a 3 hr incubation in control oocytes, tRNA export was complete, whereas about 80% of the DHFR mRNA and 60–70% of the histone H4 mRNA, U1, and U5 snRNA had moved to the cytoplasm (Figure 1A, lanes 4–6). In oocytes injected with full-length M or with its N-terminal fragment (1–77), U snRNA export was strongly inhibited and mRNA export was reduced, but tRNA export was not affected (Figure 1A, lanes 7–9 and 13–15). M fragments 51–229 and 73–229 had no inhibitory effect (Figure 1A, lanes 10–12, and data not shown). The full-length M inhibited RNA export independently

Figure 1. The N-Terminal Domain of VSV M Is Sufficient for Nuclear Export Inhibition (A) Purified recombinant proteins indicated above the lanes were injected into Xenopus oocyte nuclei together with a mixture of radiolabeled RNAs. This mixture consisted of DHFR mRNA, histone H4 mRNA, U1⌬Sm, U5⌬Sm, U6⌬ss, and human initiator methionyl tRNA. RNA samples from total oocytes (T), cytoplasmic (C), and nuclear (N) fractions were collected 3 hr after injection, or immediately after injection (t0, lanes 1–3) and analyzed on 8% acrylamide/7 M urea denaturing gels. (B) Purified recombinant proteins indicated above the lanes were injected into Xenopus oocyte cytoplasm. Following a 1 hr incubation, the mixture of labeled RNAs described in (A) was injected into oocyte nuclei. Export was allowed to take place for 3 hr. Symbols are as in (A). (C) Xenopus laevis oocyte nuclei were injected with recombinant M and a mixture of in vitro transcribed 32P-labeled RNAs consisting of U6⌬ss RNA, U1⌬Sm RNA, and Ad-CTE pre-mRNA. RNA samples were collected 3 hr after injection or immediately after injection (t0) as indicated. Products of the splicing reaction were resolved on 10% acrylamide/7 M urea-denaturing gels. The mature products of the splicing reaction are indicated diagrammatically on the left of the panel. The closed triangle represents the CTE. In all panels, one oocyte equivalent of RNA, from a pool of 10 oocytes, was loaded per lane.

of the site of injection (Figure 1B, lanes 4–6). In contrast, fragment 1–77 inhibited RNA export significantly only when injected into the nucleus (Figure 1A versus 1B, lanes 13–15), suggesting that when injected into the cytoplasm, this fragment could not access a nuclear target. Figure 1C shows that under the conditions in which

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Figure 2. Substitution of M Residues 52–54 by Alanines Abolishes Its RNA Export Inhibitory Activity (A) Positions of the alanine scan mutants are shown on the primary amino acid sequence of VSV M. Residues important for the inhibitory effect of M in RNA export are located around position 52. The position of the M51R mutation is indicated by a circle while the APPPY motif implicated in budding is underlined. (B) Purified recombinant M mutants indicated above the lanes were coinjected into Xenopus oocyte nuclei with the mixture of radiolabeled RNAs described in Figure 1A. RNA samples were collected 3 hr after injection, or immediately after injection (t0, lanes 1–3), and analyzed as indicated in Figure 1A. (C) Xenopus oocyte nuclei were injected with zz-M or zz-M(D). Protein samples from total oocytes (T), cytoplasmic (C), and nuclear (N) fractions were collected 4.5 hr after injection, or immediately after injection (t0), and analyzed by Western blot. (D) Human 293 cells were transfected with a mixture of plasmids encoding ␤-gal, CAT, and either GFP alone or C-terminally fused to M or M(D). Cells were collected 36 hr after transfection and ␤-gal or CAT activity was determined. Data from three separate experiments were expressed as the percentage of the activities measured when GFP alone was coexpressed. The data are means ⫾ standard deviations. Similar results were obtained in experiments performed with different preparations of plasmid DNA. Protein expression levels were determined by Western blot using anti-GFP antibodies.

M strongly inhibited export of U1⌬Sm RNA and of a spliced mRNA, the export of an excised intron lariat bearing the constitutive transport element (CTE) of simian retrovirus-1 was not affected (Figure 1C, lanes 7–9 versus 4–6). Similarly, as reported by Her et al. (1997), M did not interfere with the export of multiple tRNA species (i.e., tRNASer, tRNAiMet, tRNALeu, and tRNATyr; Figure 1 and data not shown). These results indicate that the inhibitory effect of VSV M in export is mediated by its N-terminal domain and is not exerted simply by blocking the central channel of the NPC. Mutation of M Residues 52–54 Abolishes Its Inhibitor Effect in Transcription and Nuclear Export To define M residues important for RNA export inhibition, we constructed alanine substitutions of residues within positions 1–77 (Figure 2A). The resulting mutants were tested by injection into Xenopus oocyte nuclei as described above. Figure 2B shows that substitution of residues 52–54 by alanines (mutant D) abolished the inhibitory effect of M in RNA export, whereas alanine scan mutants A, B, C, and E retained the inhibitory activity. The nucleocytoplasmic distribution of mutant M(D) was similar to that of wild-type M following a 4.5 hr incubation (Figure 2C), suggesting that mutant M(D) is properly folded. Thus, residues important for VSV M export inhibition are located around position 52. Previously, a methionine-to-arginine substitution at position 51 (M51R) was shown to render M protein defective in shutting off host RNA synthesis (Ferran and LucasLenard, 1997; Ahmed and Lyles, 1998). Prompted by these observations, we tested the effect of mutant M(D) on the expression of various reporter genes in a cotransfection assay. Human 293 cells were cotransfected with the reporter genes cat (chloramphenicol acetyltransferase) and lacZ (␤-galactosidase) together with pEGFPN3

plasmid, either without insert or encoding M and M(D) proteins C-terminally fused to green fluorescence protein (GFP). Western blot analysis revealed that the expression levels of M were reproducibly lower than those of M(D), as M inhibits its own expression (Black and Lyles, 1992). Furthermore, M protein inhibited expression of both cat and lacZ reporter genes, as previously reported (Black and Lyles, 1992), while coexpression of mutant M(D) had no effect (Figure 2D). Since this mutant also lacks export inhibitory activity, we conclude that mutation of the same residues of M relieves the inhibition of both transcription and export.

Wild-Type M, but Not the M(D) Mutant, Localizes to the Nuclear Envelope Next, we compared the subcellular localization of M with that of the M(D) mutant fused to GFP in transfected HeLa cells. Both the wild-type protein and the M(D) mutant distributed evenly between the nuclear and cytoplasmic compartments (Figures 3A and 3E). A fraction of M, but not of M(D), was also detected in a rim around the nuclear periphery. When transfected HeLa cells were permeabilized with digitonin prior to fixation, the nucleoplasmic and cytoplasmic pools of M were solubilized, but the fraction associated with the nuclear rim was resistant to detergent extraction (Figure 3B). The nuclear rim–associated fraction of M colocalized with the labeling produced by the monoclonal antibody 414 directed against nucleoporins (compare Figure 3B with 3D), indicating that M associates with the NPC. In contrast, in cells transfected with the M(D) mutant, the GFP signal disappeared on digitonin extraction (Figure 3F). Thus, wild-type M localizes to the nuclear envelope while mutant M(D), which is unable to inhibit transcription and export, was not detected at the nuclear rim.

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Figure 3. A Fraction of VSV M Localizes to the Nuclear Rim (A–H) HeLa cells were transfected with pEGFPN3 plasmid derivatives expressing GFP fusions of M or the M(D) mutant. In (B), (D), (F), and (H), HeLa cells were extracted with digitonin prior to fixation. In (C), (D), (G), and (H), HeLa cells expressing M-GFP or M(D)-GFP were double labeled with the monoclonal antibody 414 directed against nucleoporins. Bar, 10 micrometers.

Identification of Nup98 as a Cellular Target of VSV M As the cellular target of M could be a nucleoporin, we fractionated solubilized nuclear envelope proteins from HeLa cells by affinity chromatography. Recombinant M and the M(D) mutant expressed in E. coli were immobilized on IgG-sepharose beads. Comparison of the pattern of proteins selected on beads coated with wildtype M versus mutant M(D) by SDS-PAGE revealed the presence of a strong band of ⵑ97 kDa in the M eluates (Figure 4A, lane 3), which was absent from the control eluates (Figure 4A, lane 4). This protein was identified by peptide mapping with a MALDI-TOF instrument. The detected peptides unambiguously identify Nup98 as the protein migrating with an apparent molecular weight of 97 kDa (Powers et al., 1995; Radu et al., 1995). The identity of Nup98 was confirmed by Western blot using anti-Nup98 antibodies (Figure 4B, lane 2). As mentioned above, mutant M(D) is unlikely to have an altered conformation as, except for Nup98, the pattern of proteins binding to immobilized M or M(D) was identical when nuclear, cytoplasmic, or nuclear envelope extracts were used (Figure 4A; data not shown). Furthermore, tubulin, which was previously reported to interact with M (Melki et al., 1994), did not discriminate between M and M(D) (Figure 4B). As Nup98 is one of the major glycosylated nucleoporins, we tested whether its binding to M could be competed with by WGA. Solubilized nuclear envelope proteins from HeLa cells were selected on immobilized M

Figure 4. Identification of Nup98 as a Cellular Target of VSV M (A) Solubilized nuclear envelope (NE) proteins from HeLa cells were incubated with IgG-Sepharose beads coated with zz-tagged M or the M(D) mutant. After extensive washes, bound proteins were eluted stepwise with 500 mM and 1 M MgCl2 as indicate above the lanes. One half of the eluted fractions was analyzed by SDS-PAGE followed by silver staining. Lane 1 shows 1/200 of the input fraction. Lanes 7 and 8 show proteins eluted from the M or M(D) coated beads with 1 M MgCl2 when the HeLa extracts were omitted. The asterisk indicates the position of Nup98. (B) Samples from a similar experiment described in (A) were analyzed by Western blot using a polyclonal antibody directed against Nup98. One tenth of the input and one half of the eluted fractions were analyzed. The lower panel shows that tubulin can be selected on beads coated with M or M(D) when HeLa S-100 extracts are used. (C) Recombinant purified zz-M (lanes 1–4) or zz-M(D) mutant (lane 5) were immobilized on IgG-Sepharose beads and incubated with solubilized NE proteins (lanes 1–5). In lanes 2 and 3, WGA was included in the binding reactions. In lanes 3 and 4, N-acetylglucosamine (GlcNac) was included. After incubation and extensive washes, bound proteins were eluted with SDS sample buffer and analyzed by Western blot using anti-Nup98 antibodies.

in the presence or absence of WGA. The presence of Nup98 in the bound fractions was detected by Western blot using anti-Nup98 antibodies. As expected, Nup98 bound to immobilized M but not to its mutant form M(D)

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(Figure 4C, lane 1 versus 5). Binding of Nup98 to M was prevented by WGA (Figure 4C, lane 2). This effect was specific as it was reversed by addition of GlcNAc (Figure 4C, lane 3); GlcNAc alone had no effect (Figure 4C, lane 4). In summary, Nup98 binds to M but not to the M(D) mutant. This binding is prevented by WGA, suggesting that the O-linked sugars may contribute to the interaction. VSV M Interacts with the FG Repeat Domain of Nup98 To determine the domain of Nup98 implicated in the interaction with VSV M, we performed in vitro binding assays. Full-length Nup98, an N-terminal fragment containing the FG repeats (residues 66–515), and a fragment comprising part of the repeats and the complete C-terminal domain (residues 222–920) were expressed in 293 cells as fusions with glutathione S-transferase (GST) (Figure 5A). The recombinant proteins were solubilized with 1% Triton X-100 and tested for binding to immobilized M. Figure 5B shows that both the full-length Nup98 and its N-terminal domain (66–515) bound to immobilized M (lanes 2 and 8). These interactions must have been specific as they were competed with by WGA (Figure 5B, lanes 3 and 9). In contrast, fragment 222–920 and also fragment 66–281 were not selected on immobilized M (Figure 5B, lane 5 and not shown). Thus, the M binding domain of Nup98 is contained within residues 66–515. This fragment encompasses most of the FG dipeptide repeats (Powers et al., 1995; Radu et al., 1995), the hRAE1/Gle2 binding site, or GLEBS-like motif (Pritchard et al., 1999), and most of the predicted glycosylation sites of the nucleoporin (Figure 5A). To determine whether the interaction between M and Nup98 was direct, Nup98 fragment 66–515 was also expressed in E. coli as a GST fusion and selected on beads coated with M or the M(D) mutant. Figure 5C shows that the bacterially expressed fragment did not interact specifically with immobilized M (lanes 5–8), even though binding reactions were supplemented with 293 cell lysates. Also, in vitro translated full-length Nup98 or fragment 66–515 failed to interact with M (data not shown). In contrast, both full-length Nup98 and fragment 66–515, translated in vitro or expressed in E. coli, were able to interact with three putative Nup98 partners: RAE1 (Pritchard et al., 1999), CRM1 (Zolotukhin and Felber, 1999), and TAP (Bachi et al., 2000) (data not shown). Thus, their failure to interact with M was unlikely to be due to a misfolding of the proteins. Since these proteins interact with M only when expressed in 293 cells, we hypothesized that M recognizes the glycosylated form of Nup98. The observation that Nup98 fragments expressed in E. coli could not interact with M even in the presence of 293 cell extracts supports the model that M binds only to glycosylated Nup98. Nup98 Mediates the Inhibitory Effect of VSV M in Cellular Gene Expression To determine whether binding to Nup98 accounts for the localization of VSV M at the nuclear envelope, we compared the subcellular localization of M-GFP in wildtype to Nup98⫺/⫺ mouse embryonic cells. The nuclear rim–associated fraction of M-GFP was visualized by

Figure 5. VSV M Interacts with the FG Repeat Domain of Nup98 (A) Full-length Nup98 and fragments 222–920 or 66–515 were expressed in 293 cells as GST fusions. Cells were lysed with 1% Triton X-100, and the presence of each protein in the lysates was detected by Western blot using a polyclonal anti-GST antibody. Schematic representation of Nup98: numbers indicate the amino acid position. The vertical bars show the position of the FG repeats. The black rectangle represents the GLEBS-like motif, and the branches the position of putative glycosylation sites. (B) Lysates from human 293 cells expressing full-length Nup98 or fragments 222–920 or 66–515 were incubated with IgG-Sepharose beads coated with purified zz-M. Binding reactions were performed in the presence (⫹) or absence (⫺) of WGA, as indicated above the lanes. After extensive washes, bound proteins were eluted with SDS sample buffer. One tenth of the inputs (lanes 1, 4, and 7) and one quarter of the bound fractions (lanes 2, 3, 5, 6, 8, and 9) were analyzed by Western blot using anti-GST antibodies. (C) Nup98 fragment 66–515 fused to GST was expressed in either 293 cells or E. coli. The corresponding cell lysates were incubated with IgG-Sepharose beads coated with zz-M or the M(D) mutant as indicated above the lanes. Binding to zz-M coated beads was performed in the presence (⫹) or absence (⫺) of WGA. Note that E. coli lysates were supplemented with lysates prepared from nontransfected 293 cells.

treating transfected cells with Triton X-100 prior to fixation. As in HeLa cells, in wild-type mouse cells M-GFP localizes evenly within the cytoplasm and nucleoplasm, and concentrates at the nuclear rim (Figures 6A and 6B). In contrast, in Nup98⫺/⫺ cells M-GFP was hardly detected at the nuclear envelope (Figures 6E and 6F). To confirm that the failure to detect M at the nuclear envelope of

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clear envelope (Figures 6C and 6D). Thus, wild-type M is predominantly recruited to the NPC by interacting with the nucleoporin Nup98. We still see a fraction of M associated with the nuclear envelope in Nup98⫺/⫺ cells (Figure 6F). This is probably due to the affinity of VSV M for membranes (Ye et al., 1994), as we observed M by immunoelectron microscopy associating with the nuclear envelope in regions devoid of NPCs (data not shown). To investigate whether Nup98 mediates the inhibitory effects of M in host cell gene expression, we designed a reporter assay to monitor transcriptional inhibition in the presence of M protein. In this assay, expression of a luciferase reporter gene depends on the expression of a fusion protein consisting of the DNA binding domain of Gal4 and the transactivation domain of VP16 (Figure 6G). Thus, M and Gal4-VP16 proteins are coexpressed, while expression of the luciferase reporter is delayed and occurs almost exclusively when M protein is already present in the cell. Therefore, in this assay, Gal4-VP16mediated luciferase expression should closely reflect the cellular transcriptional activity under the influence of M. Control and Nup98⫺/⫺ embryonic cells were cotransfected with a mixture of three plasmids encoding the Gal4-VP16 fusion protein, M protein, and luciferase reporter. In control cells, coexpression of M and Gal4-VP16 inhibited luciferase activity in a gene dosage–dependent fashion (Figure 6H, control) with 37-fold inhibition when the M plasmid was cotransfected at a 1:2 ratio to the luciferase plasmid. In contrast, in Nup98⫺/⫺ cells, coexpression of M with the luciferase reporter did not reduce luciferase activity substantially, even at the highest M:luciferase plasmid ratio (Figure 6H). The expression levels of M were similar in both cell types (Figure 6H). Together, these results strongly suggest that Nup98 is the cellular factor that mediates the inhibitory effect of VSV M in the expression of cellular RNAs.

Figure 6. Nup98 Is the Major Binding Site for VSV M at the NPC (A–F) Wild-type or Nup98⫺/⫺ mouse cells were transfected with pEGFPN3-M. Nup98⫺/⫺ cells were also cotransfected with pEGFPN3-M and a plasmid encoding Nup98 (C and D). The GFP signal was detected throughout the nucleoplasm and cytoplasm (A, C, and E). A nuclear rim staining was also detected in the wild- type cells (A) or the knockout cells coexpressing Nup98 (C). In (B), (D), and (F), transfected cells were extracted with Triton X-100 prior to fixation. (G) Schematic representation of the transactivation assay. (H) Wild-type or Nup98⫺/⫺ cells were cotransfected with a mixture of plasmid DNA encoding the luciferase reporter, a Gal4-VP16 fusion protein, and VSV M. The data from three separate experiments were quantitated and expressed as fold inhibition relative to the luciferase activity in the absence of M. The data are means of three experiments ⫾ standard deviations. M expression was analyzed by Western blot.

Nup98⫺/⫺ cells resulted from the lack of Nup98, M-GFP was transfected into Nup98⫺/⫺ cells that ectopically express human HA-tagged Nup98. Restoration of Nup98 expression results in recruitment of M-GFP to the nu-

VSV M Inhibits Host Cell Gene Expression Primarily by Blocking Nuclear Export The experiments described above strongly suggest that VSV M inhibits host gene expression by targeting a nucleoporin, thereby interfering with nuclear export. To determine whether the transcription inhibition by M occurs upstream or downstream of the export block, we analyzed the effect of M protein expression on the bulk of polyadenylated RNAs by in situ hybridization. HeLa cells expressing M protein, but not the mutant M(D), accumulated polyadenylated RNA within the nucleus (Figure 7A versus 7C). If M were inhibiting transcription prior to export, a decrease in the poly(A)⫹ signal would have been observed. To confirm further that inhibition of transcription by M occurs downstream of the export block, we analyzed the transcription and export of U1⌬Sm RNA in Xenopus oocytes. Genes encoding U1⌬Sm were injected into the nuclei of oocytes, and recombinant M or mutant M(D) proteins were injected into the cytoplasm. At different time intervals, (␣-32P)GTP was injected into the cytoplasm. After a further 2 hr incubation, oocytes were dissected, and RNA from nuclear and cytoplasmic fractions or from total oocytes was analyzed. In control oocytes and in oocytes injected with mutant M(D) (Figure

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interaction with Nup98. Interfering with nuclear export by targeting a nucleoporin points to a novel strategy by which cytoplasmic RNA viruses can inhibit host cell gene expression, and represents a novel mechanism for cytotoxicity.

Figure 7. VSV M Inhibits Host Cell Gene Expression Primarily by Blocking Nuclear Export (A–D) HeLa cells expressing wild-type M or mutant M(D), C-terminally fused to GFP, were double labeled for poly(A) RNA. (E) Oocytes were injected with U1⌬Sm gene followed by recombinant M or M(D) proteins and ␣-32P-GTP as indicated diagrammatically on the top panel. RNA was analyzed as in Figure 1. Bands indicated by asterisks on the edge of the figure correspond to endogenous transcripts.

7E, lanes 1–6), U1⌬Sm transcripts were present almost exclusively in the cytoplasmic fractions (lanes 2 and 5). In contrast, in oocytes injected with wild-type M (Figure 7E, lanes 7–9), the U1 transcripts were retained in the nucleus, indicating that M protein was blocking nuclear export efficiently. Unexpectedly, M failed to inhibit transcription of U1, various endogenous transcripts (indicated by asterisks in Figure 7E), and a tRNA gene (data not shown). Since the basic transcriptional machinery in Xenopus is very unlikely to differ from that of mammalian cells, we conclude that VSV M does not directly target the transcription apparatus. Discussion The matrix protein of vesicular stomatitis virus (VSV M) shuts off host cell gene expression by inhibiting both transcription and nucleocytoplasmic transport. In this study, we show that the nucleoporin Nup98 is critical for the inhibitory effect of M on host cell gene expression. Furthermore, we demonstrate that the primary effect of VSV M is to inhibit nuclear export; its effect on transcription is thus seen to be an indirect consequence of its

A Fraction of VSV M Localizes to the Nuclear Rim M was previously shown to be evenly distributed within the cell or specifically localized at the plasma membrane (Lyles et al., 1988; Blondel et al., 1990; Ye et al., 1994; Harty et al., 1999). Its subcellular localization appeared to be subject to variation depending on the fixation procedure utilized and the cell type in which the protein was expressed (Peeples, 1988). We analyzed the subcellular localization of M fused to GFP in various cultured cell lines. The GFP tag allowed us to detect the fusion protein directly in living cells, avoiding possible artifacts from the fixation procedure. In fixed or unfixed cells, M distributes evenly within the nucleoplasm and cytoplasm. Moreover, a fraction of the protein localized at the plasma membrane and at the nuclear envelope. We are confident that the M-GFP fusion reflects the localization of wild-type M in human cells for the following reasons. First, similar results were obtained using an N-terminal zz-tagged version of M in two human cell lines: HeLa cells and U-2 OS cells (data not shown). Second, M-GFP induced cytopathic cell rounding in transfected HeLa, U-2 OS, and 293 cells. Third, M-GFP inhibited the expression of reporter genes in 293 cells and mouse cells. Thus, the GFP tag did not interfere with the cytotoxic effects or with the inhibitory activity of the protein (Blondel et al., 1990; Black and Lyles, 1992). VSV M Interferes with Nuclear Export by a Mechanism Distinct from Inhibiting Nup98 Function It is striking that microinjection of anti-Nup98 antibodies or of recombinant M in Xenopus oocytes leads to the same pattern of export inhibition. Both inhibit U snRNA export strongly, partially reduce mRNA export, and have no effect on the export of tRNA (Her et al., 1997; Powers et al., 1997; Figure 1). However, while the anti-Nup98 antibody did not inhibit protein import, M was reported to inhibit protein import when expressed in Xenopus oocytes by microinjection of its mRNA (Her et al., 1997). We have not been able to detect protein import inhibition by microinjecting recombinant M under conditions in which U snRNA export was strongly inhibited. In our experimental conditions, M was injected in the cytoplasm 1 hr prior to the injection of the reporter proteins. In the experiments reported by Her et al. (1997), oocytes expressing M were incubated for 22 hr prior to the injection of the karyophilic proteins. These oocytes accumulate importin-␣ within the nucleus. Thus, it is possible that protein import inhibition by M was a secondary consequence of the block of importin-␣ nuclear export. Consistent with this, we observed that coinjection of M with labeled importin-␣ within oocyte nuclei partially inhibits export of importin-␣ to the cytoplasm (data not shown). Thus, VSV M inhibits export mediated by CRM1, CAS, and the mRNA export receptor(s), but does not directly interfere with protein import or with export mediated by exportin-t. Moreover, we have shown that M did

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not inhibit TAP-mediated export of CTE-bearing intron lariats. M binding to Nup98 may competitively inhibit the interaction with some of its putative partners such as CRM1 (Zolotukin and Felber, 1999), TAP (Bachi et al., 2000), and hRAE1/Gle2p (Pritchard et al., 1999), thereby interfering with its function. However, the possibility that M acts solely by disrupting Nup98 function is incompatible with the observation that Nup98⫺/⫺ cells do not display an overt export inhibition phenotype (Kasper et al., 2000). Although we cannot rule out that in the Nup98⫺/⫺ cells a compensatory mechanism bypasses Nup98 function, our data support a model in which M interferes with nuclear export by a mechanism distinct from inhibition of Nup98 function. It is likely that anti-Nup98 antibodies act in a similar way. Nup98 has been localized at the baskets of the NPC (Radu et al., 1995; Fontoura et al., 1999). Studies on the export of the Balbiani ring mRNP from Chironomus tentans indicate that the nuclear baskets with their terminal rings are dynamic structures that can open when large RNPs are translocated (Kiseleva et al., 1996). Thus, M and anti-Nup98 antibodies bound to the nuclear baskets via Nup98 may interfere with the dynamic behavior of these structures. The Link between Transcription and Transport Inhibition by VSV M The Xenopus oocyte system allowed us to investigate the effects of M in RNA export independently of its inhibitory effects on transcription. This is in contrast with studies using transient transfections in cultured cells, in which the ability of M to inhibit the expression of a reporter gene is measured. In this case, the effects of M on mRNA transcription and export cannot be dissociated. Using the oocyte system, we showed that inhibition of RNA export by M is rapid and direct, as it can be observed by coinjecting the recombinant protein together with the labeled RNAs within the nucleus. In an early study, Fresco et al. (1987) showed that the effects of M on U snRNP processing and assembly, which are likely to reflect an export block, were extremely rapid and observed prior to the onset of transcription inhibition. This raised the possibility that inhibition of transcription by M was a secondary consequence of an export block. Three sets of experiments demonstrate that the effect of M on transcription is downstream of the export inhibition. First, in Nup98-deficient cells M fails to inhibit the expression of a luciferase reporter. Second, expression of M in HeLa cells results in accumulation of polyadenylated RNA within the nucleus. Third, although M strongly inhibits export in Xenopus oocytes, no inhibition of transcription by polymerase II and III was observed. Since the basic transcriptional machinery in Xenopus is very unlikely to differ from that of mammalian cells, we interpret these results as an indication that M does not target the transcription apparatus directly. The effects of M on transcription in mammalian cells may be explained by a higher turnover of transcription factors compared to the oocyte system, and thus a requirement in mammalian cells to export mRNA-encoding labile transcription factors. It is also possible that by blocking export, M interferes with some regulatory mechanism that does not exist in the oocytes.

Thus, the primary effect of M is the inhibition of nuclear export. Taking into account the growing list of cellular proteins whose activity is regulated at the level of nucleocytoplasmic transport (reviewed by Kaffman and O’Shea, 1999), it is reasonable that a block in nuclear export might lead to the pleiotropic effects associated with VSV M expression. Experimental Procedures Plasmids and Protein Expression in E. coli cDNAs encoding the M protein of VSV (Indiana serotype, San Juan strain) or the deletion mutants were amplified by PCR and cloned within the NcoI-BamHI sites of vector pQE60zz. This vector is a derivative of pQE60 (Qiagen) and has a zz tag inserted within the EcoRI and BglII restriction sites. Thus, proteins expressed using pQE60zz vector have a zz tag at their N termini and a hexahistidine tag at their C termini. M cDNA was also cloned into the NcoI-BamHI sites of pBSpAlter, a derivative of pALTER-Ex1 (Promega). For expression in human cells, VSV M cDNA was excised from the pQE60zz or the pBSpAlter vectors as an EcoRI-BamHI fragment and inserted into the EcoRI-BamHI sites of the pEGFPN3 vector (Clontech). Expression of the resulting constructs in human cells generates an M-GFP fusion with or without a zz tag at the N terminus, respectively. Alanine substitutions were introduced using an oligonucleotidedirected in vitro mutagenesis system from Stratagene (Quickchange site-directed mutagenesis) following the instructions of the manufacturer. Mutants were generated in the pQE60zz-M vector. Mutations were confirmed by restriction mapping and by sequencing. For expression in human cells, full-length Nup98 cDNA was inserted within the BamHI-NotI sites of pEBG vector. The pEBGNup98-222-920 was generated by digestion and religation of plasmid pEBG-Nup98 with BamHI-KpnI. Nup98 fragment 66–515 cloned into pGEX-4T vector was kindly provided by Dr. Francoise Stutz. This fragment was subcloned into the pEBG vector for expression in 293 cells. E. coli BL21(DE3) pLysS (Stratagene) strain was used for expression of proteins in the pGEX-4T vector and M15 pRP4 strain (Qiagen) for expression of proteins in the pQE60zz vector. Proteins were purified on glutathione agarose beads (Sigma) or Ni⫹-NTA sepharose fast flow (Qiagen) as described by Gru¨ter et al. (1998). Xenopus laevis Oocyte Microinjections and RNA Analysis DNA templates for in vitro RNA synthesis of Ad-CTE pre-mRNA, DHFR mRNA, histone H4 mRNA, U5⌬Sm, U1⌬Sm, and U6⌬ss RNA, and human methionyl tRNA have been previously described (Saavedra et al., 1997). Oocyte injections and analysis of microinjected RNA by denaturing gel electrophoresis and autoradiography analysis were performed as previously described (Hamm and Mattaj, 1990). The concentration of recombinant proteins in the injected samples was 1 mg/ml. Isolation of protein from oocytes and SDSPAGE was carried out as described (Kambach and Mattaj, 1992). DNA Transfection, CAT, ␤-Galactosidase, and Luciferase Assays Human 293 cells were transfected using the calcium phosphate method. To test the effect of M on lacZ and cat gene expression, cells were cotransfected with a mixture of plasmid DNA. This mixture consisted of 2 ␮g of pCH110 (Pharmacia), 0.5 ␮g of pcDNA3-CAT (Invitrogen), and 2 ␮g of pEGFP-N3 without insert or encoding C-terminal GFP-tagged versions of M or M(D). Cells were harvested 36 hr posttransfection. CAT activity was measured as described by Morency et al. (1987). Gal4-VP16 was tested for transcriptional activation properties on the Gal4-responsive reporter construct G5B/ pGL2 essentially as described in Kasper et al. (1999). Expression and Purification of GST-Nup98 Fusion Proteins in Human 293 Cells Human 293 cells were transfected with either pEBG-Nup98(1–920), pEBG-Nup98(222–920), or pEBG-Nup98(66–515) using the calcium phosphate method. Approximately 40 hr after transfection, cells were collected, washed once with ice-cold PBS containing 1 mM

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PMSF, and lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8.0], 0.5 mM EDTA, 1% Triton X-100, 1 mM PMSF, 1 ␮g/ml leupeptin, and 1 ␮g/ml aprotinin) for 15 min in ice. The lysate was then cleared by centrifugation at 13,000 RPM for 15 min at 4⬚C. Protein expression levels were determined by Western blot using anti-GST antibodies. Antibodies A polyclonal anti-GST serum was obtained by immunizing rabbits with bacterially expressed GST. A polyclonal antiserum to human Nup98 was kindly provided by Dr. Aurelian Radu. Monoclonal antibody mAb414 to nucleoporins was purchased from BAbCO. Immunofluorescence in HeLa Cells and Nup98⫺/⫺ Mouse Cells HeLa cells were transfected with either pEGFPN3-M or M(D) using FuGene. Mouse cells were transfected with pEGFPN3-M using Superfect. Approximately 20 hr after transfection, cells were either fixed with 3.7% formaldehyde for 10 min and subsequently permeabilized with 0.5% Triton X-100, or extracted first with 0.1% digitonin or Triton X-100 for 1 min and then fixed in formaldehyde. Doublelabeling experiments and in situ hybridizations with oligo U were performed essentially as described previously (Calado et al., 2000). Solubilization of Nuclear Envelope Proteins About 2.5 ⫻ 109 HeLa cells were lysed according to Dignam et al. (1983) to obtain nuclear and cytoplasmic extracts. After nuclear lysis, the insoluble pellet containing chromatin and nuclear envelope proteins was resuspended in 2 ml of ice-cold buffer containing 0.1 mM MgCl2, 2 mM CaCl2, 5 ␮g/ml microccocal nuclease (Calbiochem), and protease inhibitors. Subsequent steps were performed as described by Fontoura et al. (1999). Binding Assays IgG-Sepharose beads (Pharmacia) were washed extensively with 1 M NaCl, 25 mM HEPES (pH 7.9). Approximately 50 ␮l of packed beads was incubated with 50 ␮g of recombinant purified zz-tagged M or M(D) in 1 M NaCl, 25 mM HEPES (pH 7.9) for 1 hr at 4⬚C. Beads were then washed extensively with NP-40 buffer (1% NP-40, 50 mM Tris-HCl [pH 8], 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2, 2 mM DTT, 150 mM NaCl, and protease inhibitors), and incubated with 1 ml of solubilized nuclear envelope proteins previously precleared with 50 ␮l of IgG-Sepharose beads. Beads were rotated for 1 hr at 4⬚C, recovered by gentle centrifugation, and washed six times with NP40 buffer containing 200 mM KCl instead of 150 mM NaCl. Bound proteins were eluted stepwise with 500 mM and 1 M MgCl2, 25 mM HEPES (pH 7.9). The eluted proteins were precipitated with 20% trichloroacetic acid (final concentration), resuspended in SDS sample buffer, and analyzed by SDS-PAGE followed by silver staining or Western blot. In the experiments shown in Figure 5, beads were incubated with lysates prepared from human 293 cells transfected with pEGB vectors expressing Nup98 full-length or fragments 222– 920 or 66–515. After washing, beads were eluted with SDS sample buffer and analyzed by SDS-PAGE followed by Western blot using anti-GST antibodies. When indicated, WGA (Sigma) and GlcNAc (Sigma) were added to the binding reactions at a final concentration of 4.5 mM and 50 mM, respectively. Protein Identification Proteins eluted from the affinity columns were analyzed on SDSPAGE followed by silver staining. Bands of interest were excised and in-gel digested with trypsin (Shevchenko et al., 1996). Of the total digest solution, 0.3 ␮l was analyzed by peptide mass mapping on a Bruker REFLEX MALDI time-of-flight mass spectrometer (Bruker-Franzen, Bremen, Germany) using the fast evaporation technique for matrix preparation (Vorm et al., 1994). Acknowledgments The technical assistance of Michaela Rode is gratefully acknowledged. We are indebted to Drs. Lionel Arnaud, Ralph Bischoff, and Nelly Pante´ for their help. We wish to thank Dr. Aurelian Radu for the kind gift of anti-Nup98 antibodies, Dr. Barbara Felber for the Nup98 cDNA, Dr. Douglass Lyles for providing M51R-mutant cDNA, and Drs. John K. Rose, Yves Gaudin, and Danielle Blondel for the

gift of VSV M cDNA. We are grateful to Maarten Fornerod, Iain W. Mattaj, and Christel Schmitt for critical reading of the manuscript and to Nicolas Ro¨ggli for graphic work. This study was supported by the German Ministry of Research and Technology (BMBF), the European Molecular Biology Organization (EMBO), the Human Frontier Science Program Organization, and the Junta Nacional de Investigac¸a˜o Cienti´fica e Te´cnologica (Program PRAXIS XXI), Portugal. Received May 11, 2000; revised September 18, 2000. References Ahmed, M., and Lyles, D.S. (1998). Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III. J. Virol. 72, 8413–8419. Arts, G.-J., Fornerod, M., and Mattaj, I.W. (1998). Identification of a nuclear export receptor for tRNA. Curr. Biol. 6, 305–314. Bachi, A., Braun, I.C., Rodrigues, J.P., Pante´, N., Ribbeck, K., von Kobbe, C., Kutay, U., Wilm, M., Go¨rlich, D., Carmo-Fonseca, M., and Izaurralde, E. (2000). The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTEbearing RNA substrates. RNA 6, 136–158. Bastos, R., Lin, A., Enarson, M., and Burke, B. (1997). Targeting and function in mRNA nuclear export of nuclear pore complex protein Nup153. J. Cell Biol. 134, 1141–1156. Black, B.L., and Lyles, D.S. (1992). Vesicular stomatitis virus matrix protein inhibits host cell-directed transcription of target genes in vivo. J. Virol. 66, 4058–4064. Blondel, D., Harmison, G.G., and Schubert, M. (1990). Role of matrix protein in cytopathogenesis of vesicular stomatitis virus. J. Virol. 64, 1716–1725. Calado A., Kutay, U., Ku¨hn, U., Wahle, E., and Carmo-Fonseca, M. (2000). Deciphering the cellular pathway for transport of poly(A)binding protein II. RNA 6, 245–256. Dignam, J., Lebowitz, R., and Roeder, R. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from mammalian nuclei. Nucleic Acids Res. 11, 1475–1589. Featherstone, C., Darby, M.K., and Gerace, L. (1988). A monoclonal antibody against the nuclear pore complex inhibits nucleocytoplasmic transport of protein and RNA in vivo. J. Cell Biol. 107, 1289–1297. Ferran, M.C., and Lucas-Lenard, J.M. (1997). The vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter. J. Virol. 71, 371–377. Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Lu¨hrmann, R. (1995). The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475–483. Fontoura, B.M.A., Blobel, G., and Matunis, M.J. (1999). A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186 kilodalton precursor generates Nup98 and the novel nucleoporin Nup96. J. Cell Biol. 144, 1097–1112. Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I.W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, 1051–1060. Fresco, L.D., Kurilla, M.G., and Keene, J.D. (1987). Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus. Mol. Cell. Biol. 7, 1148– 1155. Gru¨ter, P., Tabernero, C., von Kobbe, C., Schmitt, C., Saavedra, C., Bachi, A., Wilm, M., Felber, B.K., and Izaurralde, E. (1998). TAP, the human homologue of Mex67p, mediates CTE-dependent RNA export from the nucleus. Mol. Cell 1, 649–659. Hamm, J., and Mattaj, I.W. (1990). Monomethylated cap structures facilitate RNA export from the nucleus. Cell 63, 109–118. Harty, R.N., Paragas, J., Sudol, M., and Palese, P. (1999). A prolinerich motif within the matrix protein of vesicular stomatitis virus and rabies virus interacts with WW domains of cellular proteins: implications for viral budding. J. Virol. 73, 2921–2929.

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